Method for LAMP (looped-mediated isothermal amplification) detection of trypanosoma evansi variant strain with deletion of ToTat1.2 gene

A technology of trypanosoma eazi and a detection method, which is applied in the biological field, can solve the problems of lack and inability to realize the detection of trypanosomiasis, and achieves the effects of short detection time, simple and fast operation, and no need for special instruments

Inactive Publication Date: 2015-02-11
ZHEJIANG ACAD OF MEDICAL SCI
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  • Abstract
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Problems solved by technology

[0005] LAMP technology has been gradually used in medical testing since 2003. At present, it is mainly used in the detection of pathogenic microorganisms and the diagnosis of infectious diseases. It has been applied to loop-mediated isothermal The patent for rapid detection of Trypanosoma evangelica by amplification technology (patent number CN 101892313 B), which targets the RoTat 1.2 variant surface glycoprotein (VSG) gene, but it is known that there are mutant strains lacking the RoTat 1.2 gene, This method cannot be used to detect the RoTat 1.2 gene deletion strain Trypanosoma evanii

Method used

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  • Method for LAMP (looped-mediated isothermal amplification) detection of trypanosoma evansi variant strain with deletion of ToTat1.2 gene
  • Method for LAMP (looped-mediated isothermal amplification) detection of trypanosoma evansi variant strain with deletion of ToTat1.2 gene

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Embodiment 1

[0021] Embodiment 1, specific DNA sequence search:

[0022] The surface glycoprotein (VSG) gene sequence of Trypanosoma evangelii variant strain JN2118Hu was retrieved from the American gene database—GENBANK, and the accession number is AJ870487.1. LAMP primers were designed for this conserved target sequence. The specific target sequence is shown in SEQ ID NO .1 shown.

Embodiment 2

[0023] Embodiment two, the design of primer

[0024] The primer design in the LAMP method is the key to the amplification of the LAMP method, and the PrimerExplorerV3 software is used to design the primers. The length of the target sequence amplified by LAMP is controlled below 300bp. LAMP requires at least 4 primers, which are the forward internal primer FIP, which consists of the F2 segment (completely complementary to the target gene F2c segment) and the F1c segment (same as the target gene 3' The F1c sequence at the end is the same); the forward outer primer F3 (completely complementary to the F3c segment of the target gene); the reverse inner primer BIP, consisting of the B2 segment (completely complementary to the B2c segment at the 3' end of the target gene) and Blc Segment (identical to the 3' end B1c sequence of the target gene); reverse outer primer B3 (completely complementary to the target gene B3c segment). The design rules for each segment are the same as for ...

Embodiment 3

[0029] Example 3. Loop-mediated isothermal amplification (LAMP) reaction

[0030] According to the principle of loop-mediated isothermal amplification (LAMP) technology, the present invention designs 4 primers for the specific fragment of the variant surface glycoprotein (VSG) gene of Trypanosoma evangelica JN2118Hu isolate, and recognizes 6 regions of the target sequence. The strand displacement property of BstDNA enzyme generates a large number of repeated stem-loop structures under isothermal conditions; the specific process is:

[0031] 1. Genomic DNA extraction of Trypanosoma evansi: The laboratory establishes an animal infection model of Trypanosoma evansi. Genomic DNA of Trypanosoma evanii was extracted using the genome extraction kit from Takara Company, A260 / A280>1.8 was analyzed by spectrophotometry, and a template was provided for the detection system, which was stored in a -80°C refrigerator for later use.

[0032] 2. Conditions for LAMP reaction:

[0033] The ...

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Abstract

The invention relates to a parasite quick detection technology in the technical field of biology, and discloses a specific primer in LAMP (looped-mediated isothermal amplification) detection of trypanosoma evansi variant strain with deletion of a ToTat1.2 gene. The specific primer comprises two pairs of LAMP primers FIP, BIP, F3 and B3 for detecting trypanosoma evansi. An LAMP method using the two pairs of primers to detect the trypanosoma evansi comprises the following steps of preparation of a reaction template, using of an LAMP reaction system and an LAMP reaction procedure, and judgment of detection results. The specific primer has the advantages that the operation is simple and convenient, the cost is low, the result is easily observed, and the specific primer is special for the detection of the trypanosoma evansi variant strain with deletion of the ToTat1.2 gene.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a specific primer used in the rapid detection technology of parasites by using the loop-mediated isothermal amplification technology to quickly detect the mutant strain of Trypanosoma evansae lacking the RoTat1.2 gene. Background technique [0002] Trypanosoma evanii ( Trypanosoma evansi ) is a hemoflagellate parasitizing in the hematopoietic viscera and blood of vertebrates. Gadflies and blood-sucking flies are the main vectors, which can cause severe trypanosomiasis in livestock such as cattle, mules, horses, sheep and dogs. Also known as Sura's disease, the main clinical manifestations are high fever, anemia, jaundice and parasitemia, and the acute case has a high mortality rate. Trypanosomiasis infection also causes severe immunosuppression in the body, often leading to other opportunistic diseases such as anthrax and Pasteurella infection. Sura disease is widely prevalent in m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12R1/90
CPCC12Q1/6846C12Q2527/101
Inventor 陆绍红陈睿童群波孔庆明郑斌楼涤丁建祖
Owner ZHEJIANG ACAD OF MEDICAL SCI
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